The renewal of the intestinal epithelium is driven by a pool of multipotential intestinal epithelial stem cells (IESCs) residing in the base of the crypts. Our preliminary studies indicate that a sub-set of post-mitotic cells in the small intestine crypt epithelium express high levels of the transcription factor, Sox9;these Sox9-expressing cells are consistent with enteroendocrine cells, and may modulate proliferative and differentiation capacity of IESCs through secreted peptides. Preliminary data demonstrates that a subset of Sox9-expressing Substance-P positive cells have a relatively 'long-lived'tenure in the adult IESC 'niche'. We provide further evidence that high Sox9-expressing cells populate the early developing gut, are the first differentiated cell type observed in developing 'mini-guts'(crypt-villus units derived from a single IESC in vitro), and are present in regenerating epithelial micro-colonies following irradiation. These observations suggest that cells marked by high levels of Sox9 are a conserved feature of the proliferating intestinal epithelium. The mechanism by which SOX9 functions in the EE population is unknown. EE cells are known to secrete both growth promoting and inhibitory peptides, so with advances made with NIDDK-K01 funding, we are now poised to study the relationships between EE cells and their influence on IESC proliferation and differentiation capacity. Our central hypothesis for this proposal is that crypt-based enteroendocrine cells (CBEE) are critical for modulating proliferation/self-renewal and differentiation of IESCs, and also for instructing crypt morphology. We extend this hypothesis to predict that Sox9 in the CBEE controls downstream target genes that extrinsically influence IESC and crypt biology. To begin to test these hypotheses we propose the following Specific Aims: 1a) To characterize CBEE cells, and determine their gene expression profile, and 1b) to indentify Sox9-dependent genes in EE cells. 2) To establish the effect of EE cells on IESC proliferation and crypt development using novel co-culture assays. 3) To generate an EE cell ablation transgenic mouse model. Successful completion of this study will: 1) establish a role for the CBEE cells, 2) identify novel EE-derived proteins involved in stem cell maintenance and differentiation, and 3) provide rationale, preliminary data and reagents for further extramural funding. The potential clinical implications of this study are identification of peptides that could be used as therapeutics to protect the IESC during chemotherapeutic or radiation treatments by slowing division rates of IESCs;or conversely identification of pro-growth peptides that could be used to enhance IESC proliferation by speeding the regenerative process following epithelium depletion cause by infection, irradiation or drug treatments. PUBLIC HEALTH RELEVANCE: A pool of epithelial stem cells drives regeneration of the intestinal epithelium during normal and injured states. Understanding the mechanisms that regulate and influence the proliferation and differentiation of the intestinal epithelial stem cell is critical for identifying therapies to enhance tissue regeneration during disease or following injury. There is evidence that a small population of cells that surround the stem cell zone in the intestinal epithelium may be providing both instructional queues for the larger structural anatomy of the intestinal epithelium, and also signals that control proliferation. We hypothesize that this population of cells is a subset of 'long-lived'hormone producing cells called enteroendocrine cells that have a critical role in intestinal stem cell biology. This study will investigate the role of these cells and active gene pathways within this subset of enteroendocrine cells to provide information regarding their role in the stem cell zone. Data gathered from this study will likely provide new mechanisms of action for these cell types and indentify novel targets for regenerative therapies.